疯草棘豆蠕孢菌吡咯啉5-羧化-还原酶基因敲除菌株的构建

发布时间：2018-08-06 17:33

【摘要】：疯草类植物广泛分布于我国西部草原,动物采食该类植物后会出现头部水平震颤、运动障碍等典型的疯草中毒症状,严重时可导致动物死亡,给我国草原畜牧业造成巨大经济损失。研究表明,疯草类植物的主要毒性成分是苦马豆素(Swainsonine,SW),而疯草棘豆蠕孢菌(Undifilum oxytropis)是疯草内苦马豆素生物合成的根本原因。目前已知,苦马豆素可由三类真菌产生,即豆类丝核菌(Rhizoctonia leguminicola)、金龟子绿僵菌(Metarhizium anisopliae)和疯草棘豆蠕孢菌。苦马豆素的生物合成通路在豆类丝核菌和金龟子绿僵菌中研究已较为深入,而有关疯草棘豆蠕孢菌苦马豆素的生物合成通路及调控合成的关键酶尚不清楚。课题组前期采用高通量测序技术对疯草棘豆蠕孢菌进行了全基因组测序,通过基因功能和注释分析并筛选出了可能的参与苦马豆素生物合成的部分关键酶,而吡咯啉-5-羧化还原酶(pyrroline-5-carboxylate reductase,P5CR)就是其中一个关键的调控酶。鉴于此,本试验以疯草棘豆蠕孢菌为研究对象,利用基因工程技术构建疯草棘豆蠕孢菌的P5CR基因敲除菌株,具体研究结果如下:1.根据疯草棘豆蠕孢菌全基因组测序结果注释的P5CR基因设计引物,以疯草棘豆蠕孢菌cDNA为模板,高保真扩增P5CR基因,PCR产物测序结果与注释的P5CR基因序列BLAST比对同源性为99%。2.以PUC19质粒为载体,构建了含潮霉素抗性基因的疯草棘豆蠕孢菌P5CR基因的敲除载体,P5CR上游目的基因序列和下游目的基因序列分别连在潮霉素抗性基因的两端。经PCR验证、测序和双酶切鉴定,成功构建载体。3.将构建好的载体质粒线性化后,利用REMI转化法将线性化载体转化原生质体,进行疯草棘豆蠕孢菌原生质体P5CR基因的敲除。接着经潮霉素筛选获得转化株,转化株提取DNA,进行PCR验证,结果显示P5CR基因敲除菌株构建成功。本研究成功获得了疯草棘豆蠕孢菌P5CR基因敲除菌株,为后续研究该基因对疯草棘豆蠕孢菌苦马豆素生物合成的影响及不产苦马豆素疯草新品种的培育奠定理论基础。
[Abstract]:The wild grass plants are widely distributed in the western grassland of our country. After feeding on this kind of plants, the animals will appear the typical symptoms such as head level tremor, dyskinesia and so on, which can lead to the death of animals. To our country prairie animal husbandry causes the huge economic loss. The results showed that the main toxic component of the plants was Swainsonine SW, and (Undifilum oxytropis) was the root cause of the biosynthesis. Up to now, it has been known that kudousuin can be produced by three kinds of fungi, I. e., (Rhizoctonia leguminicola), mycelia, Metarhizium anisopliae (Metarhizium anisopliae), and C. spp. The biosynthesis pathway of kussinin has been studied deeply in Rhizoctonia legume and Metarhizium anisopliae, but the biosynthesis pathway and the key enzyme of controlling the biosynthesis are not clear. The whole genome was sequenced by high-throughput sequencing technique in the early stage of the study. Some key enzymes involved in the biosynthesis of koumarin were screened through gene function and annotation analysis. Pyrrolidine-5-carboxylation reductase (pyrroline-5-carboxylate reductase P5CR) is one of the key regulatory enzymes. In view of this, the P5CR gene knockout strain was constructed by genetic engineering. The results are as follows: 1. A primer was designed based on the P5CR gene annotated from the whole genome sequencing results of Echinococcus spp. The results of high-fidelity amplification of P5CR gene and BLAST sequence of the annotated P5CR gene showed that the homology was 99. 2. 2. Using PUC19 plasmid as vector, the knockout vector P5CR upstream target gene and downstream target gene sequence of P5CR gene containing hygromycin resistance gene were constructed and linked to the two ends of hygromycin resistance gene, respectively. The vector was successfully constructed by PCR, sequencing and double enzyme digestion. After the plasmid was linearized, the linearized vector was transformed into protoplast by REMI transformation method, and the P5CR gene of the protoplast was knockout. Then the transformed strain was screened by hygromycin, the transformed strain was extracted from the transformed strain, and the PCR was verified. The results showed that the P5CR gene knockout strain was successfully constructed. In this study, the P5CR gene knockout strain was successfully obtained, which laid a theoretical foundation for the further study of the effect of the gene on the biosynthesis of C. spp.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q93;Q78

【相似文献】